In the last twenty years, gram-negative bacteria have become the leading agents of fatal bacterial infections in hospital patients. Each year nosocomial (hospital-acquired) bacteremia develops in approximately 194,000 patients in U.S. hospitals; of these about 75,000 die. Maki, D. G., (1981) in Nosocomial Infections (Dixon, R. E., Ed.), pp. 183-196, Yorke Medical Books, U.S.A. In this recent epidemiological review of nosocomial infections, it was reported that six major gram-negative bacilli accounted for most etiological agents; they are: Escherichia coli, Pseudomonas aeruginosa, Proteus, Klebsiella, Enterobacter, and Serratia.
At present, antibiotics are the chief weapons in combating the nosocomial infection. However, antibiotic therapy does not seem to lower the death rate very significantly for gram-negative bacteremia. Braude et al., (1977) J. Infect. Dis. 136, S167-173. The shortcomings of antibiotics might be attributed to the impermeability of the outer membrane of the gram-negative bacteria to the drugs and to their inability to counteract the lethal shocks caused by bacterial endotoxin.
In the last decade, several groups have attempted to develop passive immunization as an alternative or supplement to antibiotics for the control of nosocomial infections. It was expected that antiserum against endotoxin could prevent and reverse the effects of the toxin and could facilitate the removal of gram-negative bacteria from the circulation by the reticulo-endothelial system.
Because the clinical picture of shock from gram-negative bacterial septicemia is identical to that induced experimentally with endotoxin, shock resulting from gram-negative bacteremia is often referred to as "endotoxin shock." This is believed to be because the endotoxin is present on the surface of the outer membrane of gram-negative bacteria, and thus is in a position to react with body fluids and to cause the same disturbances as those seen after injection of endotoxin.
The endotoxins of gram-negative bacteria are lipopolysaccharides (LPS). There are at least three major antigenic regions in endotoxins. Luderitz et al., (1982) Curr. Top. Membr. Transp. 17, 79-151. Theoretically each is a target for a protective antibody or anti-endotoxin. The three antigenic regions of LPS are lipid A, core polysaccharide and O-specific polysaccharide (also referred to as O-specific chain or simply O-antigen). A schematic representation of LPS is shown in FIG. 1A. The O-specific polysaccharide vary markedly with each species and serologic type of bacteria. The lipid A and core polysaccharide of most gram-negative bacteria, however, share similar, if not identical, structures. This is especially true of the region on either side of the core-lipid A junction. This area of LPS virtually always contains phosphate, 2-keto-3-deoxy-D-manno-octonate (KDO) and D-glucosamine, and usually contains L-glycerol-D-manno-heptose (see FIG. 1B).
In rough strains of bacteria, the O-specific polysaccharide is lost through a mutation that deprives the bacteria of either the enzymes required to synthesize the O-antigen or the enzyme required to attach them to the core. Ziegler and co-workers exploited this genetic change to develop conventional (polyclonal) antisera against the uncovered core region on the assumption that antibody to the core of LPS would react uniformly with the endotoxins of all gram-negative bacteria because their core antigens are similar. Ziegler et al., (1973) J. Immunol. 111, 433-438. In order to produce antibody to core glycolipid, they prepared a vaccine from a rough mutant of E. coli 0111:B.sub.4, known as J5. This E. coli mutant has a similar LPS chemotype (core carbohydrate chain length) to that of S. minnesota Rc LPS (FIG. 1C). The rabbit antiserum obtained after immunization was designated J5 antiserum. During the past eight years this group has shown that J5 antiserum can prevent the toxic actions of endotoxins from various gram-negative bacteria and protect against lethal bacteremia in immunosuppressed animals.
Using a similar approach, McCabe and colleagues showed that rabbit antisera to Re rough mutant of Salmonella minnesota (FIG. 1C) protected granulocytopenic rabbits against lethal bacteremia, and protected mice against lethal challenge with heterologous endotoxins. McCabe et al., (1977) J. Infect. Dis. 136, S161-166. In a separate study, a polyvalent human gamma-globulin against Pseudomonas aeruginosa protected mice against the lethal infections. This antisera elicited almost no cross protection. Fisher, M. W., (1977) J. Infect. Dis. 136, S181-185.
Very recently anti-core glycolipid of the LPS was prepared by vaccinating human subjects. Zeigler et al., (1982) N. Eng. J. Med. 307, 1225-1230. When the antisera was administered to gravely ill bacteremic patients, the death rate from bacteremia was virtually halved as compared to controls. Among patients in profound gram-negative shock, the rate of recovery rose from 24% in controls to 54% in those treated with the antiserum. Preliminary data indicated that the human antiserum used for prophylaxis could reduce the fever, morbidity and bacteremia in neutropenic patients with gram-negative infections.
However, the protection mechanism of the anti-LPS serum demonstrated in the aforementioned reports remains poorly understood. In some cases the opposite effect was observed. Davis et al., (1969) J. Immunol. 102, 563-572, found, for example, that rat antiserum to LPS induced lethal hypersensitivity to endotoxin in mice, while in the same report they found that rabbit antisera to LPS lowered the death rate from endotoxin. In summary, the observed protective effect could be variable, depending on the composition and titer of the conventional antisera used, the exact strain of bacteria tested, the route of antigen or antibody administration, and the protocols used in serotherapy.
To circumvent the variable efficacy of conventional antisera in the serotherapy, Young et al., (1982) Clin. Research 30, 522a, prepared monoclonal antibodies using S. minnesota R595 LPS as the immunogen. They were found to be only modestly protective against a single species of gram-negative bacteria in their animal model of bacteremia.
Although lipid A moiety of LPS is known to be necessary for the endotoxin activity, the exact antigenic determinant(s) on the lipid A responsible for the toxicity has remained obscure. Luderitz et al. supra.